Stimulation Of Cyclic AMP Accumulation Research Articles

Hormonal stimulation of cyclic AMP accumulation and glycogen phosphorylase activity in calcium-depleted hepatocytes from euthyroid and hypothyroid rats.

Activation of glycogen phosphorylase by hormones was examined in hepatocytes isolated from euthyroid and hypothyroid female rats and incubated by Ca2+-free buffer containing 1 mM-EGTA. Basal glycogen phosphorylase activity was decreased in Ca2+-free buffer. However, the activation of hepatocyte glycogen phosphorylase, in the absence of extracellular Ca2+, in response to adrenaline, glucagon or phenylephrine was slightly lower, whereas that by vasopressin was abolished. The activation of glycogen phosphorylase by phenylephrine, adrenaline or isoproterenol (isoprenaline) in hepatocytes from euthyroid rats incubated in the absence of Ca2+ was not accompanied by any detectable increase in total cyclic AMP. The log-dose/response curves for activation of phosphorylase by phenylephrine or low concentrations of adrenaline were the same in hepatocytes from hypothyroid as compared wit euthyroid rats, whereas the response to isoproterenol was greater in hepatocytes from hypothyroid rats. However, the increases in total cyclic AMP accumulation caused by adrenaline or isoproterenol were greater in hepatocytes from hypothyroid rats than in hepatocytes from euthyroid rats. The increases in cyclic AMP accumulation caused by adrenaline or isoproterenol in Ca2+-depleted hepatocytes from hypothyroid rats were blocked by propranolol, a beta-adrenergic antagonist. In contrast, propranolol was only partially effective asan inhibitor of the activation of glycogen phosphorylase by phenylephrine or adrenaline in hepatocytes from hypothyroid rats and ineffective on phosphorylase activation in cells from euthyroid rats. These data indicate that the alpha-adrenergic activation of glycogen phosphorylase is not affected by the absence of extracellular Ca2+, and the extent to which total cyclic AMP was increased by adrenergic amines did not correlate with glycogen phosphorylase activation.

Follicular fluid depression of ovarian granulosa cell adenylyl cyclase activity

Previous studies have demonstrated that fluid from small (1–2 mm) porcine follicles is inhibitory of LH stimulation of cyclic AMP accumulation, progesterone secretion and morphological luteinization of granulosa cells from large (6–12 mm) porcine follicles. The present study measured adenylyl cyclase in granulosa cells following 2-day incubations in either the inhibitory culture medium containing 50% fluid from small follicles or in luteinization permissive media: 50% fluid from large follicles or 50% porcine serum. Basal, LH, epinephrine and NaF stimulation of adenylyl cyclase activity were all significantly reduced in the cells incubated with fluid from small follicles as compared to cells incubated in the control media. The data presented in this paper, in conjunction with previously published data suggest that the inhibitory fluid from small follicles, alters both receptor-cyclase coordination and adenylyl cyclase activity.

ADENOSINE REGULATES VIA TWO DIFFERENT TYPES OF RECEPTORS, THE ACCUMULATION OF CYCLIC AMP IN CULTURED BRAIN CELLS

Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β‐adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.

Hormonal control of oocyte meiosis, ovulation and luteinization in mammals

The ovulatory process can be regarded as a series of biochemical and morphological changes ultimately leading to the release of a mature oocyte and the transformation of the Graafian follicle into the corpus luteum. This process involves acute changes in steroidogenesis, resumption of oocyte meiosis, and finally rupture of the follicular wall and luteinization of the granulosa cells. Normally, all of these changes are induced synchronously by the pre-ovulatory LH surge. Experimentally, however, these changes in steroidogenesis, oocyte maturation and follicular rupture can be dissociated from each other showing that the LH effect is mediated via different cellular messengers. The gonadotrophins act in an orderly sequence to induce follicular maturation. The granulosa cells increase their number of LH receptors and respond to LH with increased stimulation of cyclic AMP accumulation and progesterone secretion. Concomitantly, they decrease in their FSH receptors and their response to FSH diminishes in terms of ability to stimulate cyclic AMP accumulation. The ovulatory process is associated with increased uptake of LH by the follicle; when granulosa cells are obtained from pre-ovulatory follicles and cultured they luteinize spontaneously. Steroid hormones modulate the actions of gonadotrophins on follicular maturation. In addition, there are non-steroidal factors in follicular fluid which regulate follicular maturation: an oocyte maturation inhibitor keeps the oocyte in meiotic arrest; a luteinizing inhibitor prevents the granulosa cells from luteinizing prior to follicular rupture; a folliculostatin inhibits FSH release from the pituitary gland. The functional activity and the lifespan of the corpus luteum depend on adequate pre-ovulatory as well as post-ovulatory gonadotrophic stimulation. Its lifespan may also be regulated by an LH binding inhibitor.

3H]Glycogen hydrolysis in brain slices: responses to neurotransmitters and modulation of noradrenaline receptors.

Abstract— Different agents have been investigated for their effects on [C3H]glycogen synthesized in mouse cortical slices. Of these noradrenaline, serotonin and histamine induced clear concentration‐dependent glycogenolysis.[C3H]Glycogen hydrolysis induced by noradrenaline appears to be mediated by beta‐adrenergic receptors because it is completely prevented by timolol, while phentolamine is ineffective. It seems to involve cyclic AMP because it is potentiated in the presence of isobutylmethylxanthine; in addition dibutyryl cyclic AMP (but not dibutyryl cyclic GMP) promotes glycogenolysis.Lower concentrations of noradrenaline were necessary for [C3H]glycogen hydrolysis (EC50= 0.5μM) than for stimulation of cyclic AMP accumulation (EC50= 8μM).After subchronic reserpine treatment the concentration‐response curve to noradrenaline was significantly shifted to the left (EC50= 0.09 ± 0.02 μM as compared with 0.49 ± 0.08 μM in saline‐pretreated mice) without modifications of either the basal [C3H]glycogen level, maximal glycogenolytic effect, or the dibutyryl cAMP‐induced glycogenolytic response.In addition to noradrenaline, clear concentration‐dependent [3H]glycogen hydrolysis was observed in the presence of histamine or serotonin. In contrast to the partial [3H]glycogen hydrolysis elicited by these biogenic amines, depolarization of the slices by 50 mM K+ provoked a nearly total [C3H)glycogen hydrolysis.

Iodide Induced Suppression of Thyrotropin-Stimulated Adenosine 3´,5´-Monophosphate Production in Cat Thyroid Slices

Cat thyroid slices were studied to investigate their responsiveness to thyrotropin stimulation of cyclic AMP accumulation. Ovine and bovine thyrotropin, in the presence of 2.5 mM aminophylline, induced a dose-dependent increase in the cyclic AMP content of cat thyroid tissue. Half-maximal stimulation of cyclic AMP accumulation was obtained at a thyrotropin concentration of 1-2 mU/ml. The maximal effect of thyrotropin was observed at 10 mU/ml, and was associated with a mean 77 +/- 19-fold increase in thyroidal cyclic AMP. Preincubation of cat thyroid tissue for 2 h with 50 micron NaI resulted in an impairment in the subsequent ability of thyrotropin to enhance cyclic AMP accumulation, without altering the level of cyclic AMP in tissues not exposed to the hormone. Preincubation alone was without effect on thyrotropin stimulation of cyclic AMP, and the inhibitory effect of iodide was prevented by addition of 3 mM methimazole to the preincubation medium. In addition, the time course of thytrotropin stimulation of cyclic AMP accumulation in cat thyroid slices was not significantly altered by the preincubation with excess iodide. These studies provide additional evidence that excess iodide inhibits the adenylate cyclase-cyclic AMP system in thyroid tissue.

Thyroxine secretion by isolated hog thyroid cells: A cyclic AMP independent pathway

The release of 131I-labeled thyroxine (T 4) from isolated hog thyroid cells was increased 1.5–2-fold by thyrotropin (TSH). Dibutyryl cyclic AMP failed to reproduce this TSH action. In this in vitro system another cell activity, T 4 synthesis, was stimulated in an essentially identical fashion by TSH and dibutyryl cyclic AMP (time course of action, dose-response relationship). 3-Isobutyl-l-methylxanthine (IBMX), 0.5 mM, did not alter the basal [ 131I]T 4 release whereas it enhanced the [ 131]T 4 synthesis. TSH, 60 mU/ml, increased the intracellular cyclic AMP concentration 3–4-fold. Chlorpromazine (5 × 10 −4 M) abolished the TSH stimulation of cyclic AMP accumulation but did not alter the TSH-induced increase in [ 131I]T 4 secretion. It is concluded that the TSH action on [ 131I]T 4 secretion by isolated thyroid cells is not mediated by the adenylate cyclase-cyclic AMP system.

Dopaminergic stimulation of cyclic AMP accumulation and parathyroid hormone release from dispersed bovine parathyroid cells.

The effects of dopaminergic agonists and antagonists have been studied in dispersed bovine parathyroid cells. Dopaminergic agonists caused a transient 20- to 40-fold increase in cellular cyclic AMP and a 2- to 3-fold increase in parathyroid hormone release. Dose-response relationships were similar for cyclic AMP accumulation and hormone release, whether studied by increasing agonist concentration or by increasing concentration of antagonist with constant agonist. The effects on the dopamine receptor could be differentiated from those of the previously characterized beta-adrenergic receptor by specific inhibitors. These results appear to represent proof with a homogeneous cell population that dopaminergic receptors linked to adenylate cyclase can regulate a secretory process mediated by cyclic AMP. This system should be useful in further studies on dopamine receptors and should provide a valid tool for determining interactions of radiolabeled ligands with such receptors.

Stimulation of prolactin synthesis and of adenosine 3':5'-cyclic phosphate formation by prostaglandins and thyroliberin in cultured rat pituitary cells

1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.

Stimulation of cyclic AMP accumulation by pentylenetetrazol

The convulsant agent, pentylenetetrazol (PTZ), increased the content of cyclic AMP (cAMP) in rat cortical slices incubated in vitro in physiologic media. This increase was partially reversed by theophylline. The addition of PTZ to maximally effective concentrations of either adenosine or 2-chloroadeosine resulted in a significantly greater than additive augmentation of cAMP accumulation, suggesting that PTZ may produce its effect by enhancing the action of endogenous adenosine. The PTZ response was not antagonized by either diphenylhydantoin, phenobarbital or ethosuximide.

Influence of tricyclic antidepressants on the adenylate cyclase-phosphodiesterase system in the rat cortex

The actions of ten tricyclic antidepressant drugs were evaluated with respect to inhibition of the adenylate cyclase-cyclic adenosine 3': 5'-monophosphate (cyclic AMP)-phosphodiesterase system in various preparations of rat cerebral cortex. At highest concentrations (10 −4M) all drugs exhibited antagonism of the stimulation of cyclic AMP accumulation by norepinephrine in incubated tissue slice (whole cell) preparations. In these experiments iprindole and desipramine (10 −4–10 −8M) were the most potent agents, while lesser degrees of inhibition were seen with amitriptyline, chlorimipramine, nortriptyline, protriptyline, imipramine, opipramol, 2-hydroxyimipramine and 2-hydroxydesipramine in approximately that order. On the other hand, in broken cellular preparations from isolated neuronal and glial-enriched fractions, extremely high concentrations (1–10 mM) of the tricyclic antidepressants were necessary to inhibit by 50% the basal, fluoride-sensitive (catalytic site) and norepinephrine-sensitive (receptor) components of adenylate cyclase. In similar studies with isolated neuronal and glial fractions somewhat lower concentrations (0.1–1 mM) of tricyclic antidepressants were required to inhibit by 50% the total phosphodiesterase activity. The data indicate a basic problem relating to drug experimentation on cyclic nucleotide systems. Results obtained from intact cellular investigations i.e. tissue slices may not always be extrapolated to similar findings with enzyme preparations derived from broken cells.

Reduced Epidermal Cyclic Amp Accumulation Following Prostaglandin Stimulation: Its Possible Role In The Pathophysiology Of Psoriasis

Since prostaglandins are known to be one of the modulators of cyclic AMP, a study of the role of prostaglandins and their relation to the regulation of the nucleotide in psoriasis was initiated. Guinea-pig epidermal preparations were incubated with prostaglandins (PGs) in the presence of theophylline. PGE1 increased cyclic AMP levels 3.5-fold whereas a 2-fold increase was seen with PGE2 and PGA1. The increase in cyclic AMP accumulation was linear with increasing concentration of PGE1, from 10(-11) to 10(-5) M. PGE1 significantly increased cyclic AMP in psoriatic epidermis in vitro; however, the stimulation of cyclic AMP accumulation was significantly less in involved epidermis as compared with uninvolved tissue. The specificity of this stimulation, its occurrence at physiologic levels, and the decreased responsiveness of the cyclic AMP system in psoriatic epidermis to PGE1 stimulation suggest that the altered response of epidermis to PGs may be one of the factors in the pathophysiology of psoriasis.

Effects of Porcine Calcitoriin on the Metabolism of Calcium and Cyclic AMP in Rat Skeletal Tissuein Vivo

Infusion of 50 mU/100 g body wt of calcitonin induced a rapid and marked increase of cyclic AMP in calvaria as well as in plasma of thyroparathyroidectomized rats. These changes preceded the decrease in plasma calcium concentration seen after hormone administration. The possible relationship of this stimulation of cyclic AMP accumulation to the hypocalcemic effect of calcitonin was examined under several experimental settings. It was found that a dose of calcitonin sufficient to cause a significant decrease of plasma calcium failed to produce any detectable accumulation cyclic AMP in calvaria, but when high doses of calcitonin were given, there was an apparent correlation between the integrated change of cyclic AMP metabolism and the duration and magnitude of hypocalcemia. Experiments employing the ophylline and imidazole, which alter cyclic AMP metabolism, led to an apparent dissociation between the hypocalcemic effect of calcitonin and its stimulation of cyclic AMP accumulation in bone.

Thyroidal autoregulation: Iodide-induced suppression of thyrotropin-stimulated cyclic AMP production and iodinating activity in thyroid cells

In continuing our study of the thyroidal autoregulation phenomenon, we have investigated the effects of iodide on several thyroidal responses to thyrotropin. Thus, we have found that the 2–4-fold thyrotropin stimulation of protein iodination in beef thyroid cells was reduced about 30% by 4 h of preincubation with 10 μM iodide, and virtually abolished with 50 μM iodide. Similarly the 8-fold thyrotropin stimulation of cyclic AMP accumulation in the cells was reduced about 30% by 3 h of preincubation with 50 μM iodide. It appears therefore that the so-called autoregulation of the thyroid gland does include influences of iodide on the thyrotropin stimulation of cyclic AMP production, iodide transport, and protein iodination which can be demonstrated in vitro in the dispersed thyroid cell system. Two other effects of thyrotropin, namely, the stimulation of [ 14C]leucine incorporation into protein and of iodide efflux were not at all affected by treatment with excess iodide, and hence may not be subject to the autoregulatory influence of iodide.

Cyclic AMP production in isolated rat seminiferous tubule cell preparations: A potential [formula omitted] assay for follicle stimulating hormone

A simple procedure for the isolation of rat seminiferous tubule cell preparations free of Leydig cells is described. Both ovine and human FSH stimulated cyclic AMP accumulation. in proportion to the concentration of the hormones. Other hormones, including ICSH and hCG, were inactive. It is proposed that stimulation of cyclic AMP accumulation in tubule cells may be a specific, sensitive and rapid in vitro assay for FSH.

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling with investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20-30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both alpha-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effectivema parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fractionmthe results suggest that these drugs are acting on a parotid acinar cell through a beta1-adrenergic mechanismmat the lowest concentrations tested, each of the adrenergic agonists stimulated significant alpha-anylase release with no detectable stimulation of cyclic AMP accumulationmeven in the presence of theophylline, phenylephrine at several concentrations increased alpha-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intra-cellular concentration of cyclic AMP may not be necessary for stimulation of alpha-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of alpha-amylase release by isoproterenol. Stimulation of alpha-amylase release by phenylephrine was only partially blocked by either alpha- or beta-adrenergic blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolaminemphenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and alpha-amylase release by N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate; These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using alpha-adrenergic blocking agents as tools for investigation of alpha- and beta-adrenergic antagonism.

Stimulation of cyclic AMP accumulation by arachidonic and 8, 11, 14-eicosatrienoic acids in rat anterior pituitary gland

The effects of fatty acids on adenosine 3',5'-monophosphate (cAMP) accumulation in rat anterior pituitary gland were studied in vitro. Of all the fatty acids tested, namely oleic, lifiolenic, γ-linolenic, cis-5-eicosaenoic, cis-8, 11, 14-eicosatrienoic, cis-11, 14, 17-eicosatrienoic, arachidonic, and cis-5, 8, 11, 14, 17-pentaenoic, only the direct precursors of prostaglandin e 1 ( cis-8, 11, 14-eicosatrienoic acid) and prostaglandin E 2 (arachidonic acid) increased intracellular cAMP levels. Stimulation was apparent within l min of incubation and reached a maximum at 15 min. At longer time intervals, the concentration of cAMP decreased to basal level. Calcium is not essential for the action of fatty acids while theophylline does not result in any qualitative change. It is concluded that fatty acids stimulate adenylate cyclase activity in rat anterior pituitary tissue and that prostaglandin synthesis is probably involved as mediator of their action.

Stimulation of cyclic AMP accumulation in the rat anterior pituitary in vitro by analogs of luteinizing hormone-releasing hormone

Pour determiner in vitro la relation structureactivite dans l'hypophyse anterieure du rat, on demontre que differents analogues de l'hormone LH-RH («luteinizing hormone-releasing hormone») ont stimule l'accumulation de l'AMP cyclique. Il semble que ces analogues exercent leur activite en stimulant l'enzyme adenyle cyclase plutot que par l'inhibition de l'enzyme phosphodiesterase.

Characteristics of Action of Prostaglandins on Cyclic AMP Accumulation in Rat Anterior Pituitary Gland

Prostaglandins (PGs) were found to lead to a marked stimulation of cyclic AMP accumulation in rat anterior pituitary gland in vitro in the following decreasing order of potency: PG E-1 E-2 GREATER THAN A-1 A-I GREATER THAN F-1ALPHA F-2ALPHA. The effect of PGs is potentiated by theophylline. The stimulatory effect of PGs on cyclic AMP accumulation is already detected 2min after the addition of 1-x 10-7 to 1-x 10-6 M PG E-2 and its maximal effect is reached after approximated 30 min of incubation, with a progressive decrease toward basal cyclic AMP levels at later time intervals. Increased intracellular cyclic AMP concentrations are accompanied by an increased release of the nucleotide into incubation medium. Complete removal of Ca-e+ from the incubation medium by addition of EGTA was found to increase the stimulatory effect of PG E-2 ON CYCLIC AMP accumulation. The action of PGs on hormonal release and cyclic AMP accumulation support the hypothesis of a role of PGs in the mechanism of anterior pituitary hormone (particularly growth hormone) release.

The effects of interstitial cell-stimulating hormone, its subunits, and recombinants on isolated rat leydig cells

The stimulation of cyclic AMP accumulation and testosterone synthesis in isolated rat Leydig cells by ovine and bovine ICSH, their subunits prepared by a new, mild procedure (dissociation of subunits at pH 3 and salt fractionation) and the recombined hormones have been studied. Whereas the isolated subunits exhibit less than 0.2% of the potency of the native hormones, recombination of the subunits results in full restoration of the biological activity. In contrast to this, recombination of the subunits prepared by a countercurrent distribution method resulted in only partial regeneration of the biological activity. The ovine hormone was found to be twice as active as the bovine ICSH. Both heterologous hybrids of the subunits of the ovine and bovine hormones were significantly more active than the bovine hormone. The utility of the isolated rat Leydig cell system as a rapid, sensitive bioassay for ICSH is also discussed.